Clinical assay for AFP-L3 by using multiple reaction monitoring–mass spectrometry for diagnosing hepatocellular carcinoma

Hyunsoo Kim, Areum Sohn, Injoon Yeo, Su Jong Yu, Jung Hwan Yoon, Youngsoo Kim

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Abstract

BACKGROUND: Lens culinaris agglutinin-reactive fraction of -fetoprotein (AFP-L3) is a serum biomarker for hepatocellular carcinoma (HCC). AFP-L3 is typically measured by liquid-phase binding assay (LiBA). However, LiBA does not always reflect AFP-L3 concentrations because of its low analytical sensitivity. Thus, we aimed to develop an analytically sensitive multiple reaction monitoring–mass spectrometry (MRM-MS) assay to quantify AFP-L3 in serum. METHODS: The assay entailed the addition of a stable isotope-labeled internal standard protein analog, the enrichment of AFP using a monoclonal antibody, the fractionation of AFP-L3 using L. culinaris agglutinin lectin, deglycosylation, trypsin digestion, online desalting, and MRM-MS analysis. The performance of the MRM-MS assay was compared with that of LiBA in 400 human serum samples (100 chronic hepatitis, 100 liver cirrhosis, and 200 HCC). Integrated multinational guidelines were followed to validate the assay for clinical implementation. RESULTS: The lower limit of quantification of the MRM-MS assay (0.051 ng/mL) for AFP-L3 was less than that of LiBA (0.300 ng/mL). Thus, AFP-L3, which was not observed by LiBA in HCC samples (n 39), was detected by the MRM-MS assay, improving the clinical value of AFP-L3 as a biomarker by switching to a more analytical sensitive platform. The method was validated, meeting all the criteria in integrated multinational guidelines. CONCLUSIONS: Because of the lower incidence of false-negative findings, the MRM-MS assay is more suitable than LiBA for early detection of HCC.

Original languageEnglish
Pages (from-to)1230-1238
Number of pages9
JournalClinical Chemistry
Volume64
Issue number8
DOIs
StatePublished - Aug 2018

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